Early and accurate assessment of suspected acute myocardial infarction is critically dependent on the sensitive and specific detection and quantitation in blood, serum or plasma of released cardiac muscle intracellular components in order to distinguish a potentially lethal event in need of emergency measures from non-life threatening conditions such as angina and non-cardiac chest pain such as dyspepsia. Early electrocardiographic changes are neither adequately specific nor sensitive, and the medical profession has come to rely on serum biochemical markers of cardiac tissue injury for early diagnosis. Initially, the serum markers creatine kinase (CK) and specifically the cardiac CK-MB isoform were used, and subsequently myoglobin as a more sensitive early indicator of cardiac damage. More recently, cardiac troponin complex and its subunits have come to be preferred as markers of myocardial damage because of their high specificity. These tests in combination, along with other markers of skeletal muscle damage, provide a high degree of diagnostic accuracy. If performed in the emergency room, an early and accurate diagnosis of myocardial damage offers great advantage to a suspected heart attack victim.
Diagnostic tests employing cardiac markers are described, for example, in U.S. Pat. Nos. 5,604,105 and 5,290,678. These and other procedures offer the rapidity of diagnosing myocardial infarction in the emergency room setting and offer significant medical benefit for patients. Diagnostic tests in which the level of troponin subunits or complexes is measured in bodily fluids frequently utilize purified troponin subunits or complexes as antigens for the preparation of antibodies used in the assay procedure, as well as the purified subunits or complex used as controls and calibrators in performing the assays. Assay calibrators are used to prepare a series of dilutions by which a standard curve across the operating range of an assay is prepared; assay controls are used to confirm that an assay is operating properly by ensuring that the assayed value of pre-determined samples fall within an acceptable range around their labeled values. In order for the assay to be calibrated properly, the troponin controls and calibrators must remain stable and in a form which is immunodetectable by the antibody.
Troponin is a muscle protein integrally involved in the calcium-dependent regulation of muscle contraction. Troponin exists in both cardiac and skeletal muscle as a non-covalently-bound complex of three subunits, the isoforms troponin C, the calcium-binding subunit, troponin I, the inhibitory subunit, and troponin T, which locates the troponin complex on tropomyosin. In vitro under the proper conditions, the troponin subunits will spontaneously associate to form non-covalently-bound complexes, e.g., troponin I and C, and troponin I, C, and T. Differences exist between the amino acid sequences of the cardiac muscle and skeletal muscle troponin isoforms.
Upon cardiac muscle injury and necrosis, troponin leaks from heart tissue into circulation, where its sensitive detection can help diagnose a heart attack. The amino acid sequence differences between the cardiac and skeletal muscle isoforms of the troponin subunits are exploited in diagnostic tests which specifically measure the cardiac isoform of the troponin subunits and complexes. Diagnostic tests for cardiac troponin I are available.
However, troponin I is inherently of poor structural stability, and is subject to proteolytic cleavage by proteases present in biological samples. A troponin I standard prepared in a bodily fluid matrix is thus subject to conformational alteration and degradation and is unsuitable as a calibrator or control. Furthermore, the inherently more stable troponin complexes are known to dissociate on storage, and thus become susceptible to proteolytic degradation. In the instance of troponin I, it must be complexed with troponin C in order to help maintain its conformational structure and stability; however, because of the nature of the affinity of the subunits in the complex, the fraction of the troponin I present in the form of a complex is concentration dependent. This limits its utility as an assay calibrator or control. The extent of interactions between the subunits may be calculated from the dissociation constant, K.sub.d [for example, as reported in Biochemistry 33:12729 [1994]]. By calculation and experimental measurement, only a limited amount of troponin I is bound to troponin C, especially over the range that would be found in patient serum samples, and thus the levels at which calibrators and controls must be used. For example, at 50 ng/ml, the upper range of most troponin I assays, only 10% of the troponin I is bound to troponin C when the two subunits are present at a ratio of 1:1. With up to 10-fold more troponin C to troponin I, and in the presence of divalent cations, a claim (Larue et al., U.S. Pat. No. 5,583,200) to the stability of the complex in the cold was minimal, i.e., "at least one day." Maintaining higher concentrations of the complex increases the degree of association; however, after dilution to the level necessary to calibrate an assay, the subunits dissociate and become immunologically unstable. Dissociation then subjects the subunits to proteolytic attack, further reducing the utility of such calibrators and controls.
Thus, need exists for stable troponin calibrators and controls to meet the needs of the industry.
Numerous troponin preparations from both natural and recombinant sources have been described that contain troponin I together with troponin C. Malnic and Reinach (1994, Eur. J. Biochem., v. 222, pp. 49-54) produced a recombinant complex in vivo by cloning all three chicken skeletal muscle troponin subunits into one or more expression plasmids. Within the expression vector each troponin gene had its own promoter, and the proteins were expressed within the bacterium as individual troponin subunits, which subsequently formed complexes within the bacterium. Fujita-Becker et al. (1993, J. Biochem., v. 114, pp. 438-444) described the reconstitution of rabbit skeletal troponin complex from recombinant subunits expressed in E. coli. None of these recombinant products has been demonstrated to have adequate stability for use as a diagnostic test standard or calibrator. As mentioned above, even the complexes of troponin subunits are not stable and will not remain bound together in solution to any great extent.
As will be evident below, a principal object of the present invention is to provide a stable troponin preparation for assay and other uses which comprises troponin I and troponin C on a single polypeptide chain, prepared as a recombinant construct and expressed in a bacterial expression system as a single polypeptide. The present invention is distinct from troponin subunits and their fragments which have been chemically cross-linked for biochemical studies using methods such as carbodiimide cross-linking and photo-crosslinking chemistry, for example as those described by Jha et al. (1996, Biochemistry, vol. 35, pp. 11026-11035), Kobayashi et al. (1996, Biochem. Biophys. Acta, vol. 1294, pp. 25-30) and Kobayashi et al. (1995, Biochemistry, vol. 34, pp. 10946-10952). In these references, specific fragments of different troponin proteins were chemically cross-linked in order to investigate the conformations of the subunits and their natural interactions in troponin complexes.
Thus, there is a need for a troponin material which meets stability requirements and of ease of preparation of purification that may be used as an antigen and as controls and calibrators among troponin assays. As there is no universally-accepted control or calibrator for troponin, it is not possible to standardize the assay between laboratories or even instruments, as each particular troponin assay along with its controls and calibrators produces results unique to that laboratory and selection of assay components. Thus, it is now impossible to provide normal and abnormal ranges that are recognized by all laboratories and physicians. These exists a need for universal calibrators and controls that can be used on all available commercial assay instruments.
It has now been discovered that a single-chain polypeptide comprising human cardiac troponin I and human cardiac troponin C is stable and has utility for the aforementioned purposes. Moreover, the product must be easily produced by the skilled artisan. This ease of production maximizes the reproducibility of the products of the invention.